Our growth regulation research has been concerned with oncogenes as positive regulators and tumor suppressor genes as negative regulators of normal and neoplastic growth. The main current project is focused on the molecular biology of the tumor suppressor gene DLC1, including the targets that regulate it and the targets that it regulates. DLC1 is inactivated frequently in a wide range of tumors, but many aspects of it mechanism of action remain poorly understood. It negatively regulates Rho, via its Rho-GAP activity, but must encode other activities, as other Rho-GAPs are not known to be inactivated in cancer. One of our main hypotheses is that DLC1 is frequently inactivated in cancer because it encodes a multifunctional protein. In support of this possibility, we have previously determined that DLC1 interacts with: 1) members of the tensin gene family, via an N-terminal region of DLC1 for which no function had been previously identified; 2) with focal adhesion kinase (FAK) and with talin, via a shared 8 amino acid motif with homology to LD motifs in paxillin near the region of the protein that binds tensin; and 3) with caveolin-1 (CAV-1), via the StAR-related lipid transfer (START) domain near the C-terminus of DLC1. Analysis of various DLC1 mutants indicated that the binding sites for each of these interactions contributed to the growth suppressor function of DLC1, but that this binding did not affect in vivo Rho-GAP activity of DLC1. These studies validate the hypothesis that DLC1 is a multifunctional protein whose biological activity depends both on its RhoGAP activity and its ability to bind a variety of signaling molecules. To examine the role of endogenous DLC1 in the control of RhoGTP and cell transformation, we inactivated DLC1 in mouse embryo fibroblasts (MEFs) by disrupting the genetically engineered endogenous floxed DLC1 alleles with an adenovirus encoding the Cre recombinase. Inactivation of DLC1 resulted in an increase in RhoGTP without cell transformation, indicating that endogenous DLC1 is limiting for the regulation of Rho, but that inactivation of DLC1 is not sufficient to drive transformation. However, additional passage of the cells with inactivated DLC1 did progress to cell transformation and anchorage-independent growth, in contrast to control MEFs that continued to express endogenous DLC1. An evaluation of genes that contributed to the progressed phenotype led to the identification of the down-regulation of p15-Ink4b and p16-Ink4a expression, and the up-regulation of CDK4 and CDK6 activity. In publicly available human tumor datasets with gene expression profiles, a poor prognosis was identified for these genes in conjunction with low DLC1 expression in lung adenocarcinoma and colorectal cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression. In addition, we have identified CDK5, which is a cytoplasmic protein whose physiologic role promotes differentiation, as a major activator of DLC1. This activation occurs because DLC1 has 4 serines that are phosphorylated by CDK5. When these serines, which are located N-terminal to the Rho-GAP domain of DLC1, are not phosphorylated, the N-terminus binds to the Rho-GAP domain, which places DLC1 in a closed, inactive conformation. When the serines are phosphorylated, it decreases the interaction of the N-terminus with the Rho-GAP domain, which places DLC1 in an open, active conformation. In cancer, CDK5 behaves as a pro-oncogenic factor, presumably because it stimulates more pro-oncogenic targets than anti-oncogenic targets, such as DLC1. Consistent with that hypothesis, down-regulation of DLC1 greatly increases the pro-oncogenic activity of CDK5 in cancer cells.